The project objective is continued development of sequence-specific crosslinking agents for nucleic acids. Such compounds when combined with an appropriate polynucleotide carrier strand allow specific blockage of any desired sequence in RNA or DNA. A variety of compounds of the form, NH2NHCO(CH2)nC(OR)2CHR'X, will be tested to determine the best length, stability, and reactivity for maximum efficiency and specificity of crosslinking. Thereafter, conditions for attachment and activation of crosslinking agent will be optimized as well as conditions for stabilization of resulting crosslinks. These studies will employ phage T7-mRNA and T7-DNA. Subsequently, a method for systematic mapping of viral genomes will be developed utilizing single-stranded T7 restriction fragments derivatized with affinity crosslinking agent. Derivatized fragments will be used for introducing a series of specific covalent blocks in the informational strand of T7-DNA. The effect of each of these blocks will be assessed by in vitro coupled transcription-translation of the blocked genomes. Compounds described herein should be valuable for in vitro studies of replication, repair, transcription, and translation. They should also enable systematic mapping and characterization of viral genomes. If in vivo applications prove feasible, the compounds will likely have important applications in the study of gene expression in procaryots and possibly in eucaryotic cells.